HODGKIN S disease is generally regarded as a

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1 Vol. No. HODGKIN S DISEASE WITH MONOCLONAL AND POLYCLONAL REED STERNBERG CELLS HODGKIN S DISEASE WITH MONOCLONAL AND POLYCLONAL POPULATIONS OF REED STERNBERG CELLS MICHAEL HUMMEL, PH.D., KATHARINA ZIEMANN, HETTY LAMMERT, STEFANO PILERI, M.D., ELENA SABATTINI, M.D., AND HARALD STEIN, M.D. Abstract Background. There is strong evidence that Reed Sternberg cells have a lymphoid phenotype, but clonally rearranged genes for B-cell and T-cell antigen receptors have not been demonstrable in tumor tissue from most patients with Hodgkin s disease. To elucidate this issue, we assayed single Reed Sternberg cells from patients with classic Hodgkin s disease of a B-cell immunophenotype to detect rearranged immunoglobulin variable-region heavy-chain ( ) genes. Methods. We isolated single Reed Sternberg cells from frozen sections that had been immunostained for CD. The rearranged genes of these cells were amplified by the polymerase chain reaction and analyzed by gel electrophoresis and nucleotide sequencing. Results. In all patients, the Reed Sternberg cells studied contained rearranged genes. Three patterns were observed: in three patients the rearrangements in HODGKIN S disease is generally regarded as a distinct type of malignant lymphoma, because Reed Sternberg cells are present in an admixture of various nonmalignant cells. Immunologic studies of Reed Sternberg cells from the nodular-sclerosing, mixed-cellularity, and lymphocyte-depleted types of Hodgkin s disease have revealed the presence of the lymphoidactivation markers CD and CD in nearly every case,, and that of B-cell or T-cell markers in a substantial proportion of cases. - These findings suggest that Reed Sternberg cells originate in activated lymphocytes of either the B-cell or T-cell type. Studies of cell lines derived from tissue affected by Hodgkin s disease give further evidence of the lymphoid nature of Reed Sternberg cells and the existence of B-cell and T-cell types. - However, studies of rearrangements of antigen-receptor genes carried out on whole-tissue DNA from biopsies of patients with Hodgkin s disease were inconclusive, because in most instances the clonal rearrangement expected in a typical lymphoma was not found. - This result may have been due to the scarcity of clonally rearranged Reed Sternberg cells or to the actual absence of a clonal rearrangement. Studies of karyotypes of cells in metaphase and interphase, DNA content, mutation patterns in the p locus, and the terminal repeats of Epstein Barr virus (EBV) genomes were similarly inconclusive because the results were heterogeneous, applicable in only some cases, or not attributable specifically to Reed Sternberg cells.,- A new approach to ascertaining the origin and clonality of Reed Sternberg cells is the analysis of single From the Institute of Pathology, Klinikum Benjamin Franklin, Freie Universität Berlin, Berlin, Germany (M.H., K.Z., H.L., H.S.); and the Hematopathology Unit, Second Service of Pathological Anatomy, University of Bologna, Bologna, Italy (S.P., E.S.). Address reprint requests to Professor Stein at the Institute of Pathology, Klinikum Benjamin Franklin, Freie Universität Berlin, Hindenburgdamm, Berlin, Germany. Supported by the Deutsche Krebshilfe, Mildred Scheel Stiftung; the Berliner Krebsgesellschaft; and a grant from the Italian Association for Cancer Research (Milan). each patient were identical, in six patients all the rearrangements were unrelated and unique, and in three patients both identical and unrelated rearrangements were detected. Apparently somatic mutations of genes were present in some Reed Sternberg cells but absent in others. Conclusions. Reed Sternberg cells with B-cell phenotypes have rearranged genes; therefore, these cells arise from B cells. The pattern of gene mutations suggests that Reed Sternberg cells can correspond to either immunologically naive or memory B cells. In half our patients the population of Reed Sternberg cells was polyclonal; in the other half, monoclonal or mixed cell populations were found. Correlation with the clinical stage suggests that polyclonal Hodgkin s disease can present as a widespread lymphoma. (N Engl J Med ; :-.) Reed Sternberg cells isolated from tissues affected by Hodgkin s disease. This approach has been used by three groups, but with differing results. - The discrepancies may be due to the small numbers of patients and subtypes of Hodgkin s disease investigated, to differences in isolation methods, or both. In this paper, we report our results with the single-cell assay in patients with Hodgkin s disease whose Reed Sternberg cells had a B-cell immunophenotype. Our method of isolating immunostained cells directly from frozen sections allows a clear morphologic and immunophenotypic identification of Reed Sternberg cells, permits the reliable collection of single Reed Sternberg cells, and prevents their contamination by other cells. METHODS Tissues Biopsy specimens from patients with Hodgkin s disease ( with the nodular-sclerosing type and with the mixed-cellularity type) containing CD-positive Reed Sternberg cells were obtained from the files in our departments. One hyperplastic tonsil specimen, one specimen of mantle-cell lymphoma, the B-cell line Raji, and the T-cell line HUT were used as control tissues and cells. Immunophenotyping, Tumor-Cell Fraction, Mitotic Index, and in Situ Hybridization Sections embedded in paraffin, frozen sections, or both were stained with antibodies against CD, CD, CD, CD, CD, CDa, EBVencoded latent membrane protein, and terminal deoxynucleotidyl transferase (TdT) from Dako (Glostrup, Denmark) and were stained for T-cell receptor b-chain (TCRb) with bf (T-cell Sciences, Cambridge, Mass.). In situ hybridization with probes for EBV-encoded nuclear RNA and (EBER and ) was performed as described elsewhere. The number of CD-positive Reed Sternberg cells among cells of other types was determined. The mitotic index of the Reed Sternberg cells was investigated by counting the number of mitotic figures in CD-positive Reed Sternberg cells. Isolation of Single Cells Frozen sections mm thick were immunostained for CD, CD, or TCRb. In addition to CD-positive and TCRb-positive cells used as controls, at least CD-positive Reed Sternberg cells were Downloaded from nejm.org on January,. For personal use only. No other uses without permission. Copyright Massachusetts Medical Society. All rights reserved.

2 THE NEW ENGLAND JOURNAL OF MEDICINE Oct., isolated from each specimen and collected as described by Küppers et al. (Fig. ). All isolations of cells were performed at least twice by different persons. Polymerase Chain Reaction A nested polymerase chain reaction (PCR) was performed with the consensus primers FR and LJH in the first round of amplification and the consensus primers FRA and VLJH for reamplification. The first PCR was carried out in the tube to which the single cell had been transferred, with ng of FR primers ( ng each), ng of LJH primer, and. mmol of magnesium chloride per liter of solution in a total volume of ml. The PCR consisted of cycles at C and cycles at C for the annealing of primers. A percent aliquot of the first amplification product was used as a template for reamplification, with ng of FRA, ng of VLJH, and. mmol of magnesium chloride per liter of solution. The second PCR consisted of cycles at C. All other PCR conditions were the same as previously described., Six microliters of each amplification product was subjected to polyacrylamide-gel electrophoresis and subsequently stained with ethidium bromide. Analysis of DNA Sequences RC A B MC Figure. Isolation of a Single CD-Positive Reed Sternberg Cell from a Section of Tissue from a Patient with Hodgkin s Disease ( ). The tissue section is shown before (Panel A) and after (Panel B) the isolation of the cell. The immunostained Reed Sternberg cell was cut away from the surrounding cells with a manipulation capillary (MC) and transferred to the reception capillary (RC) without damage to surrounding cells and tissue, as described by Küppers et al. The isolation of amplified products and subsequent analysis of DNA sequences were performed as described elsewhere. The sequences obtained were compared with each other and with published sequences of immunoglobulin variable-region heavy-chain ( ) germ lines (GenBank, release ) and translated into protein. Sequences with substitutions of more than three bases were regarded as somatically mutated, because there is very little polymorphism in the germline sequences. Control Experiments RESULTS The PCR method was capable of detecting identical gene rearrangements in single Raji cells, a culture of monoclonal B cells (Fig. A), whereas DNA from single cells of the T-cell line HUT could not be amplified with the gene primers. Approximately percent of single B cells isolated from frozen sections of hyperplastic tonsils and a mantle-cell lymphoma yielded PCR products. The PCR assay revealed unrelated (polyclonal) gene rearrangements in the tonsillar B cells and identical (clonal) rearrangements in the mantle-cell lymphoma cells. Single T cells and buffers that covered the frozen sections during the cell-isolation procedure yielded no amplification products (Fig. A). PCR and Sequence Analysis of Single Cells Isolated from Hodgkin s Disease Tissues The material analyzed by PCR from biopsy specimens of tissues affected by Hodgkin s disease included single CD-positive Reed Sternberg cells and, as positive and negative controls, single B cells and T cells, respectively. Whereas T cells yielded no amplification products, gene specific PCR products were obtained from to percent of B cells and about percent of single Reed Sternberg cells. The failure to obtain such products from the rest of the cells was probably due to the use of tissue sections, which often contain only parts of nuclei, especially in the case of large Reed Sternberg cells. It proved impossible to increase the yield by increasing the thickness of the sections, which only reduced the quality of immunostaining and made the identification and isolation of single cells less reliable. PCR products of genes were obtained from individual Reed Sternberg cells isolated from all patients with Hodgkin s disease. Gel electrophoresis and analysis of gene sequences revealed three patterns (Table and Fig. B). In three patients (Patients,, and ), all the amplification products of Reed Sternberg cells from a given biopsy specimen had the same length and sequence. In six patients (Patients through ), the lengths and gene sequences of the amplification products from each biopsy specimen differed. In the remaining three patients (Patients,, and ), some PCR products had the same lengths and sequences, whereas other products had different ones. Each experiment was performed at least twice by two persons, with identical results in each case. Comparison of Sequences and Translation into Protein We compared the gene sequences we studied with germ-line sequences in the GenBank data bank to identify individual genes and infer the presence of mutations in Reed Sternberg cells (Table, and material on deposit with the National Auxiliary Publications Downloaded from nejm.org on January,. For personal use only. No other uses without permission. Copyright Massachusetts Medical Society. All rights reserved.

3 Vol. No. HODGKIN S DISEASE WITH MONOCLONAL AND POLYCLONAL REED STERNBERG CELLS A bp B bp B Cells T Cells Raji Buffer S S Patient Patient Patient S S and, in variable quantity and density, the B-cell marker CD. Early lymphoid-cell markers, such as CDa, CD, and TdT, were not detected (data not shown). The Reed Sternberg cells of all four patients with nodular sclerosing Hodgkin s disease contained unrelated (polyclonal) gene rearrangements and lacked detectable transcripts of EBER and. The average mitotic index of the Reed Sternberg cells was significantly lower (. percent) in the polyclonal group, with late mitotic figures almost totally absent in three instances, as compared with the monoclonal group (mitotic index,. percent). Service [NAPS]*). The population of genes that the Reed Sternberg cells had rearranged resembled the population used by normal B cells. Mutations of genes varied greatly, from none to sequences that appeared to be highly mutated within the same patient and between different patients. One patient (Patient ), with a polyclonal population of Reed Sternberg cells, had only wild-type sequences. The DNA sequences of the genes from the Reed Sternberg cells were potentially translatable into protein, and were thus functional, with the exceptions of a deletion of base pairs (in Patient ) that resulted in a frame shift, and of three other sequences, each in a different patient, in which the translation broke off in the N region of the rearranged gene. Correlation of Rearrangement Patterns with Other Features The histologic, immunophenotypic, and other features shown in Tables and indicate that the Reed Sternberg cells from all the patients expressed CD *See NAPS document no. for three pages of supplementary material. Order from NAPS c/o Microfiche Publications, P.O. Box, Grand Central Station, New York, NY -. Remit in advance (in U.S. funds only) $. for photocopies or $ for microfiche. Outside the U.S. and Canada, add postage of $. ($. for microfiche postage). There is a $ invoicing charge for all orders filled before payment. Figure. Amplification Products of the Genes Generated by PCR with the Use of the FRA and VLJH Primers. Panel A shows amplification products after percent polyacrylamide-gel electrophoresis and staining with ethidium bromide. The products were derived from single selected normal B cells (lanes through ) and cytospin preparations of single cells from the Raji cell line (lanes and ). No amplification products were obtained from single selected T cells (lanes through ) or overlying buffers (lanes and ). Panel B shows -specific amplification products derived from single Reed Sternberg cells isolated from tissue from three patients with Hodgkin s disease. In Patient (lanes through ), all the PCR products differed in length; in Patient, products of both identical length (lanes and ) and different lengths (lanes and ) were found, whereas in Patient (lanes through ) all the PCR products were the same length. S denotes a molecular-weight standard, and bp base pairs. Downloaded from nejm.org on January,. For personal use only. No other uses without permission. Copyright Massachusetts Medical Society. All rights reserved. DISCUSSION We conducted a PCR analysis of the rearranged genes in single Reed Sternberg cells with B-cell immunophenotypes that were isolated from patients with classic Hodgkin s disease. We focused on this B-cell type of Reed Sternberg cells, because with whole-tissue DNA we found a striking positive correlation between the expression of the B-cell marker CD on Reed Sternberg cells and the presence of clonal gene rearrangements. However, analysis of whole-tissue DNA cannot determine whether the clonal rearrangements are derived from Reed Sternberg cells or from other cells in the biopsy specimens. Therefore, we turned to the analysis of individual Reed Sternberg cells. With this approach, we found rearranged genes in Reed Sternberg cells in all patients studied. Our results, and the demonstration of immunoglobulingene rearrangements in single Reed Sternberg cells in two other studies,, suggest that technical factors may have contributed to the failure of Roth et al. to obtain PCR products from single Reed Sternberg cells in any of their patients. By molecular means, our experiments show the B-cell origin of Reed Sternberg cells that express the B-cell antigen CD. The pattern of gene rearrangements in the patients we studied was heterogeneous. In three patients, all the Reed Sternberg cells isolated from the same biopsy specimen had identical rearrangements. This result is consistent with the molecular findings in a monoclonal B-cell lymphoma. We were surprised, however, by the unrelated rearrangements in other patients. The rearranged genes of some Reed Sternberg cells were identical, whereas those of the others in the same tissue sample were unrelated. These findings indicate a polyclonal proliferation of Reed Sternberg cells in six patients and mixed populations of both monoclonal and polyclonal cells in the other three patients. Typically, the B cells in a reactive lymph node

4 THE NEW ENGLAND JOURNAL OF MEDICINE Oct., consist of a polyclonal population, whereas in a lymphoma the B cells are monoclonal. The heterogeneous pattern of gene rearrangement can explain most of the differing results of previous studies using DNA that was extracted either from tissue samples or from enriched populations of Reed Sternberg cells, as well as the heterogeneous findings of analyses of the DNA content of Reed Sternberg cells., The reproducibility of our results diminishes the possibility that they represent methodologic artifacts. Nevertheless, our data seem to be at variance with the results obtained by several other groups. In the study of single cells by Delabie et al., only polyclonal populations of Reed Sternberg cells were detected, whereas Küppers et al. found only monoclonal tumor cells. This discrepancy is probably due to the small numbers of patients three and four, respectively included in these two studies. A study of the DNA of Reed Sternberg cells has been interpreted to indicate that the cells have a monoclonal origin. However, it is possible that some of the patients in that study, especially those without aneuploidy, chromosomal aberrations, or p mutations, had Reed Sternberg cells of polyclonal origin. Most studies of EBV genomes in Hodgkin s disease have found evidence of monoclonal EBV episomes with a molecular probe of the terminal-repeat region of the virus. However, this probe has also found monoclonal EBV episomes in some samples of hyperplastic (polyclonal) lymphoid tissue and in some cases of HIVrelated immunoblastic lymphomas with polyclonal populations of rearranged immunoglobulin genes. The rearranged genes in the three patients with Table. Analysis of Single Reed Sternberg Cells from Patients with Classic Hodgkin s Disease with a B-Cell Immunophenotype, Performed to Detect Gene Rearrangements. REARRANGEMENT PAT- TERN AND PATIENT Polyclonal Mixed polyclonal and monoclonal Monoclonal AMPLIFICA- TION PRODUCTS SEQUENCES GENE FAMILY* WITH TOTAL IDENTICAL LENGTHS TOTAL *Sequences were compared with known germ-line sequences in a data bank (GenBank, release ). Numbers following a slant line are the numbers of identical sequences. A difference of nucleotides or less from the germ-line sequence in the data bank was considered to represent a low degree of deviation; a difference of to nucleotides, a medium degree; and a difference of more than nucleotides, a high degree. As revealed by percent polyacrylamide-gel electrophoresis. Differences in the length of the amplification products could not be determined, but the sequences differed. monoclonal gene rearrangements that we studied had somatic mutations. In each patient, all the Reed Sternberg cells isolated had the same distinctive mutations. The heterogeneous pattern of the gene mutations in five of the six patients with polyclonal Hodgkin s disease indicates that in a given tissue DEGREE OF DEVIATION FROM KNOWN SEQUENCES* IDENTICAL LOW MEDIUM HIGH / / no. of sequences / / / / / /; deletion of bp with frame shift / Table. Clinical, Histologic, and Immunohistologic Features of Patients with Classic Hodgkin s Disease Whose Reed Sternberg Cells Were Positive for CD and CD and Negative for CD and T-Cell Receptor.* *NS denotes nodular-sclerosing type, MC mixed-cellularity type, and EBV Epstein Barr virus. According to the Ann Arbor classification system of disease stages. As determined by staining with antibodies against latent membrane protein and by in situ hybridization with EBER probes. Plus signs denote the presence of CD-positive cells, minus signs the presence of CD- negative cells, and both signs together the presence of both CD-positive and CD-negative cells. No cells in this sample were in anaphase or telophase. PATIENT / AGE (YR)/ SEX / / DISEASE STAGE DISEASE SUBTYPE EBV- POSITIVE REED STERNBERG CELLS % OF ALL CELLS % IN MITOSIS CD /F IIA NS No.. /M IIIA NS No.. / /F IIA NS No.. /M IIeA NS No.. /M IVA MC Yes.. /M IIA MC Yes.. / /M IIA MC Yes.. /M IIIB MC No.. /F IIA MC No.. /F IVB MC Yes.. /F IIIB MC Yes.. /M IIB MC Yes.. sample the Reed Sternberg cells were not only unrelated, but also probably derived from B cells in different stages of maturation; those without somatic mutations could correspond to B cells that had not yet entered the germinal center, whereas those with gene mutations may have originated from memory B cells that had left the germinal center. If the Reed Sternberg cell is indeed the neoplastic component of Hodgkin s disease, then our finding of polyclonal Reed Sternberg cells conflicts with current concepts of tumorigenesis. Monoclonal neoplasms grow through continuous mitotic divisions and give rise to identical progeny. In the case of polyclonal Reed Sternberg cells, the mechanism of cell growth must differ. Quantitative and qualitative analyses of mitosis indeed revealed that the mitotic index was far lower in the patients with polyclonal cells than in those with monoclonal cells. Moreover, three of the six patients with polyclonal Reed Stern- Downloaded from nejm.org on January,. For personal use only. No other uses without permission. Copyright Massachusetts Medical Society. All rights reserved.

5 Vol. No. HODGKIN S DISEASE WITH MONOCLONAL AND POLYCLONAL REED STERNBERG CELLS berg cells, but none of the three with monoclonal Reed Sternberg cells, lacked late mitotic figures, suggesting a disturbance of the mitotic process, of cytokinesis, or both. These observations suggest that polyclonal populations of Reed Sternberg cells arise from the continuous recruitment of unrelated B lymphocytes. Such a mechanism would be predicated on the susceptibility of certain B cells to be transformed into Reed Sternberg cells (a process perhaps mediated by genetic instability); a transforming agent or agents, such as EBV; and an immune defect that impairs the elimination of aberrant cells. There is evidence of each of these elements in Hodgkin s disease. - The classification of Hodgkin s disease as polyclonal or monoclonal may have clinical implications. For example, the patients with polyclonal Reed Sternberg cells may respond better to chemotherapy than those with monoclonal Reed Sternberg cells. Our study shows that the presence of B-cell markers on Reed Sternberg cells does not constitute an example of aberrant gene expression, but indicates a real relation between those cells and B cells. We therefore conclude that there are B-cell types of Hodgkin s disease and that some of them contain polyclonal populations of Reed Sternberg cells. Further studies of single cells may clarify the origin of Reed Sternberg cells that express T-cell antigens or of those that lack both B-cell and T-cell antigens. We are indebted to B. Kalvelage, C. Kreschel, and H.-H. Müller for excellent technical assistance; to Ioannis Anagnostopoulos, M.D., for valuable assistance with the Discussion section, and to Joannah Caborn for her editorial assistance. This paper comprises part of the doctoral thesis of Ms. Ziemann. 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6 THE NEW ENGLAND JOURNAL OF MEDICINE Oct.,.Kapp U, Wolf J, Hummel M, et al.hodgkin s lymphoma-derived tissue serially transplanted into severe combined immunodeficient mice. Blood ;:-..Barrios L, Caballin MR, Miro R, et al.chromosome abnormalities in peripheral blood lymphocytes from untreated Hodgkin s patients: a possible evidence for chromosome instability. Hum Genet ;:-..Klitz W, Aldrich CL, Fildes N, Horning SJ, Begovich AB.Localization of predisposition to Hodgkin disease in the HLA class II region. Am J Hum Genet ;:-..Mack TM, Cozen W, Shibata DK, et al.concordance for Hodgkin s disease in identical twins suggesting genetic susceptibility to the young-adult form of the disease. N Engl J Med ;:-..Diehl V, Pfreundschuh M, Loffler M, et al.chemotherapy of Hodgkin s lymphoma with alternating cycles of COPP (cyclophosphamide, vincristin, procarbazine, prednisone) and ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine): results of HD and HD trials of the German Hodgkin Study Group. Med Oncol Tumor Pharmacother ;:-. Downloaded from nejm.org on January,. For personal use only. No other uses without permission. Copyright Massachusetts Medical Society. All rights reserved.